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Abstract

Background

The cause of sporadic amyotrophic lateral sclerosis (ALS) is largely unknown but hypotheses
about disease mechanisms include oxidative stress, defective axonal transport, mitochondrial
dysfunction and disrupted RNA processing. Whereas familial ALS is well represented
by transgenic mutant SOD1 mouse models, the mouse mutant wobbler (WR) develops progressive
motor neuron degeneration due to a point mutation in the Vps54 gene, and provides an animal model for sporadic ALS. VPS54 protein as a component
of a protein complex is involved in vesicular Golgi trafficking; impaired vesicle
trafficking might also be mechanistic in the pathogenesis of human ALS.

Results

In motor neurons of homozygous symptomatic WR mice, a massive number of endosomal
vesicles significantly enlarged (up to 3 μm in diameter) were subjected to ultrastructural
analysis and immunohistochemistry for the endosome-specific small GTPase protein Rab7
and for amyloid precursor protein (APP). Enlarged vesicles were neither detected in
heterozygous WR nor in transgenic SOD1(G93A) mice; in WR motor neurons, numerous APP/Rab7-positive
vesicles were observed which were mostly LC3-negative, suggesting they are not autophagosomes.

Conclusions

We conclude that endosomal APP/Rab7 staining reflects impaired vesicle trafficking
in WR mouse motor neurons. Based on these findings human ALS tissues were analysed
for APP in enlarged vesicles and were detected in spinal cord motor neurons in six
out of fourteen sporadic ALS cases. These enlarged vesicles were not detected in any
of the familial ALS cases. Thus our study provides the first evidence for wobbler-like
aetiologies in human ALS and suggests that the genes encoding proteins involved in
vesicle trafficking should be screened for pathogenic mutations.

Background

Neurodegenerative processes cause dramatic but heterogeneous disease phenotypes depending
on the onset of symptoms, disease progression and the particular type of neurons being
affected. In the case of "Amyotrophic Lateral Sclerosis" (ALS) the motor neurons of
the motor cortex, brain stem and spinal cord are affected. The degeneration of motor
nerves causes denervation of skeletal muscle and progressive muscular weakness leading
eventually to paralysis and death. Despite intensive research no effective therapeutic
treatment is available but recently some progress has been made in the understanding
of the underlying molecular mechanisms of ALS.

The majority of neurodegenerative disorders are associated with abnormal protein aggregation.
Aggregates of amyloidogenic cleavage products of amyloid precursor protein (APP) are
implicated in the pathogenesis of Alzheimer disease (AD, recently reviewed by [1]). APP accumulation also occurs within intracellular vesicles in Niemann Pick Disease
type C (NPC) [2] and recently found to be elevated in skeletal muscles of ALS patients as well as
SOD1-G93A mutant transgenic mouse [3]. For these reasons we elected to investigate APP accumulation in the wobbler mouse,
another animal model for human ALS with a different pathomechanism.

The recessive wobbler mutation (wr = gene symbol, phenotype WR) spontaneously occurred 50 years ago in the breeding stock
of Falconer [4] and was later mapped to the proximal mouse chromosome 11 [5]. Homozygous (wr/wr) wobbler mice develop the first disease symptoms at the age of three to four weeks.
From this time point onwards the muscle weakness, beginning in the forelegs, proceeds
to death. The life expectancy of homozygous WR mice is around 120 days. Degeneration
of WR motor neurons is accompanied by activation of glia cells (reactive gliosis and
microglia activation) and shows striking similarities to early-onset ALS cases. The
phenotype of the wobbler mouse has been intensively investigated throughout the last
50 years and studies towards potential therapies have been conducted. A positional
cloning of the wobbler gene revealed a highly conserved vesicle trafficking factor, Vps54 [6]. The wobbler mutation is a point mutation leading to an amino acid replacement (Q967L) in the C-terminal
domain of Vps54 causing the ALS-like motor neuron degeneration. We also demonstrated
that the complete loss of Vps54 function leads to embryonic lethality around day 11.5
of embryonic development [6].

Yeast, as well as mammalian Vps54 forms a complex with Vps52 and Vps53, called GARP
(Golgi associated retrograde protein) complex [7,8], which is required for tethering and fusion of endosome-derived transport vesicles
to the trans-Golgi network (TGN) [9]. The GARP complex tethers vesicles to their target membrane (TGN) leading to a t-SNARE
- v-SNARE dependent fusion of the membranes. In mammalian cells, Vps52 interacts with
the tSNARE Syntaxin10 and the small GTP binding protein Rab6 [8]. In wobbler mice, mutation of leucine-967 to glutamine causes instability of an alpha
helical structure, leading to reduced levels of Vps54 and consequently, the GARP complex
[9]. In this study we characterise abnormally enlarged endosomal vesicles in wobbler
spinal cord motor neurons and provide evidence that similar vesicles can be found
in motor neurons of a subset of human ALS cases. These findings provide evidence for
morphological criteria that could be used to classify human MND.

Results

Fine structure of WR motor neurons

To analyse the consequence of impaired vesicle trafficking in WR motor neurons, light
(Figure 1A&B) and electron microscopy (EM, Figure 1C-J) of spinal cord sections from symptomatic wobbler (42 d.p.n.) and age-matched unaffected
controls (heterozygous wr/+) was performed. In wobbler spinal cord, a small fraction of motor neurons is symptomatic
for neurodegeneration, judged by morphological changes ranging from early vacuolisation
to obvious neuronal cell death (Figure 1A&1B). Degeneration stages were classified based on Nissl staining as described earlier
[10]. Though nuclear morphology changes from distinct rounded contours to slightly irregular
ones (Figure 1A&1C), no change in nuclear morphology, as characteristic for apoptosis or necrosis can
be observed, even at later stages of degeneration. Changes at the ultra structural
level are transparent enlarged vesicles / vacuoles which are derived from the Golgi
apparatus (Figure 1G&1H). In more advanced stages of degeneration, these vacuoles increase in size and become
more frequent (Figure 1I). A second type of vacuoles, according to their position and their association with
ribosomes, appear to originate from dilatation of the endoplasmic reticulum (ER),
as demonstrated in earlier stages (postnatal day 7 to 14) of the wobbler disease [11]. At terminal stages, motor neuron degeneration is characterized by extensive vacuolisation
(Figure 1A, C, E, J) with vacuoles graded from smaller up to extremely large (2-3 μm diameter) ones (Figure
1C, E, J). Initially, areas with strong vacuolisation seem to be localized in one region of
the cell (Figure 1C, E) but later they are found all over the motor neuronal soma (Figure 1A) obviously sparing the axon and the axon hillock. No difference in number and distribution
of lysosomes was observed between wobbler and wild-type motor neurons (Figure 1C, D, E, F).

As demonstrated earlier, the small GTPase protein Rab7 has a role in late endosomal
pathway and in the final maturation of autophagic vesicles [12]. The wobbler mutation causes impaired retrograde transport of vesicles. Thus we speculated
that Rab7 might be expressed differently in wobbler motor neurons. Spinal cord sections
from unaffected heterozygous (genotype wr/+) and wobbler (WR, genotype wr/wr) mice were stained for Rab7 (Figure 2B, E) and revealed enlarged vesicles in wobbler motor neurons compared to wild-type controls.
Interestingly, staining for APP, an abundant membrane protein, which is transported
via the secretory pathway, showed an identical localisation as Rab7 preferentially
in endosomes (Figure 2A, D), indicating that APP and Rab7 are co-localised in enlarged vesicles in wobbler motor
neurons (Figure 2F). This type of co-localisation was only seen in degenerating motor neurons, whereas
morphologically normal motor neurons showed no such abnormalities. In addition, spinal
cord motor neurons from symptomatic SOD1 tg (G93A) mice (120 d.p.n.) exert a vesicular
distribution of APP and Rab7 distinct from the observations made in WR motor neurons
(Figure 2G, H, J). The enlarged vesicles characterised by EM and by APP immunohistochemistry (IHC)
were subjected to image analysis to quantify cross-sectional vesicular diameters in
wildtype, wobbler, and SOD1 tg (G93A) mice (Figure 3). A significant difference was seen in endosomal diameters in wild-type and wobbler
motor neurons, both by EM and by IHC to a similar extent (0.5 μm vs. 2.5 μm, and 0.8
vs. 3 μm, respectively), whereas the endosomes in SOD1 tg (G93A) motor neurons were
only slightly enlarged (1 μm vs 0.8 μm in WT, p < 0.001), supporting the notion that
large endosomal aggregates are indicative for the WR disease with impaired vesicle
trafficking.

Figure 2.APP and Rab7 colocalise in degenerating motor neurons. Representative immunohistochemical staining for Amyloid-Precursor Protein (APP)
(A,D,G) using rabbit anti-APP, and for Rab7 (B,E,H) using goat-anti-Rab7, and co-localisation
of both proteins (C,F,I) in spinal cord motor neurons of 42 d.p.n. wild-type (WT,
A,B,C), age-matched wobbler (WR, D,E,F), and 120 d.p.n. SOD1G93A (G,H,I) transgenic
mice. Note the spotty appearance of the large vesicular structures with up to 2.5
μm diameter in WR sections. These structures were not seen in WT and in SOD1 (G93A)
transgenic spinal cord sections. Bar in A, valid for A-I, 5 μm.

Distinct localisation of APP and LC3 in degenerating motor neurons

To test the hypothesis that enlarged vesicles in WR motor neurons could be autophagosomes,
co-stains of APP and LC3 were performed in a representative motor neuron with normal
(WT, Figure 4A, B, C) and with degenerating morphology (WR, Figure 4D, E, F). In both motor neuron populations, LC3-positive vesicles were indistinguishable
in size and number between WT and WR, and most of APP-positive large vesicles in wobbler
motor neurons did not stain for LC3 and vice versa (Figure 4C, F). We conclude that the abnormally enlarged vesicles in WR motor neurons are distinct
from autophagosomes.

Figure 4.APP/LC3 immunohistochemistry in WR motor neurons. Immunostaining for APP (A,D; green) using rabbit-anti-APP and mouse-anti-LC3 (B,E;
red) performed in either WT (A,B) or WR (D,E) motor neurons in cervical spinal cord
(42 d.p.n.). Note a similar number of autophagosomes in B and E (arrowheads). Arrows
in D indicates large APP-positive vesicles. Merged images in C and F demonstrate that
APP and LC3 are localised to distinct vesicles in degenerating motor neurons. Bar
in A, valid for A-E, 10 μm.

Analysis of APP processing in brain stem and spinal cord of wild-type and wobbler
mice

Accumulation of APP could be due to abnormal secretase processing. Due to a distinct
expression of APP in motor neurons in the spinal cord (see Figure 2) we analysed brain stem and spinal cord protein extracts from WT and WR mice for
expression levels of APP, LC3 and Rab7 (Figure 5). Significant increases in APP, LC3 and Rab7 protein levels were detected in WR brain
stem and spinal cord lysates compared to WT. No APP processing products, however,
were detected, suggesting that accumulation of APP is not due to abnormal processing
in WR motor neurons. The levels of LC3 expression is higher in WR spinal cord, however
the ratios of LC3I (18kD) and LC3II (16kD) were unchanged, indicating that autophagosome
activation is not affected. For Rab7 protein, a slight increase in WR spinal cord
was also observed, concomitant with the observation of endosome accumulation in WR
motor neurons (Figure 2). With respect to APP processing, we stained WT and WR spinal cord sections for the
secretases ADAM10 and BACE, but neither a difference in distribution nor in concentration
was detected (data not shown).

Figure 5.APP processing in WR spinal cord. Western blot analysis of brain stem (BS) and spinal cord (SC) lysates from age-matched
WT and WR mice. Total lysates (20 μg per lane) were separated on an 8% polyacrylamide
gel, blotted and subjected to immunostaining using 22C11 (APP), LC3 and Rab7 as described.
The positions of APP695 and APP770, of LC3I (18kD form) and LC3II (16 kD form) and
Rab7 (22 kD) are marked on the left. Anti-tubulin antibody (lower panel) was used
as loading control. Band intensities for APP695, LC3II and Rab7 were quantified from
3 independent experiments and plotted as ratio WR/WT ± SEM. For each WT/WR pair, the
WT value was set to 1.

APP-positive enlarged vesicles in human MND/ALS autopsies

From these previous results we concluded that the presence of enlarged APP-positive
vesicles could be indicative for motor neuron diseases with impaired retrograde protein
transport. We analysed a collection of human MND autopsy samples with well-documented
case histories but unknown aetiologies for the presence of APP positive enlarged vesicles
after immunohistochemistry (Figure 6 and Table 1). From fourteen documented MND cases investigated, six MND cases displayed enlarged
vesicles in spinal cord motor neurons. Two familial MND cases with mutations in the
SOD gene did not exert enlarged vesicles, which could match our findings in SOD1(G93A)
motor neurons and implies that SOD1 mutations are distinct in motor neuron degeneration
mechanisms from the sporadic ALS cases with impaired trafficking that we investigated
here. Motor neurons with enlarged vesicles were found in individual motor neurons
in the spinal cord of autopsies with ALS or Motor neurons disease (MND), a representative
example of this staining is shown (Figure 6). Analyses of APP immunoreactivity in MND patients in optical z- and y-axes revealed
that APP is present in vesicle structures with diameters of 1.5-4 μm (average 2.8
μm), very similar to the dimensions of enlarged APP-positive vesicles observed in
WR motor neurons.

Figure 6.APP staining in the spinal cord of human ALS autopsies. Representative (case No. 12 see table 1 for details) degenerating human motor neuron
with enlarged APP-positive vesicles in the cell soma. For comparison, a morphologically
normal spinal cord motor neuron from the same autopsy is shown above (arrow). (A),
a single plane image, (B) and (C), maximum projections from a stack of images obtained
from confocal optical sectioning. (A), low magnification of MND spinal cord section
stained with anti-APP. (B), high power magnification of the normal MN shown in (A),
as a xy- (middle), yz- (left) and xz- (top) scan with diffuse staining of APP in the
cell soma; (C), high power magnification of the degenerating MN shown in A, as an
xy- (middle), yz- (left) and xz- (top) scan with enlarged vesicles, some of them larger
than 2 μm (see crossing point of the planes in C with a vesicle diameter of 2.8 μm.
The yz- and xz-scans reveal the three-dimensional APP-positive vesicles in the degenerating
human motor neuron. Scale bar in (A), 50 μm.

Sequence analysis of Vps54 gene of ALS patients

Genomic DNAs of all six ALS patients with wobbler-like enlarged endosomal structures
as well as controls were prepared from cerebellum samples. All exons of human VPS54
were amplified including flanking regions, and sequenced in both orientations. In
none of the samples and none of the VPS54 exons a sequence variation could be detected,
thus, no mutation or polymorphism could be found in the VPS54 gene of the selected
ALS patients.

Discussion

Our study demonstrates that impaired retrograde transport as observed in wobbler motor
neurons is characterized by the presence of enlarged endosomal vesicles with positive
immunostaining for Rab7 and APP, but not for LC3. As such, the type of motor neuron
degeneration as observed in WR does not involve autophagosomes. These findings contrast
those for motor neurons of SOD1(G93A) mice where extensive autophagosome activation
has been demonstrated [13]. It is unlikely that the vesicular accumulation of APP is instrumental for motor
neuron degeneration in the wobbler mouse, as APP transgenic mice crossed into the
wobbler mutation did not enhance motor neuron pathology (Schmitt-John et al, unpublished
observations).

Characteristic signs of motor neuron death in wobbler are enlarged vesicles blebbing
of the Golgi apparatus, increasing in size and number in later stage as well as enlarged
vesicles/vacuoles derived from the ER, in agreement with earlier EM studies performed
on wobbler motor neurons [11]. With the exception of this vacuolisation, the affected motor neuron seems to be
viable just up to the terminal breakdown. The ultrastructural data indicate that the
vesicular shuttle system of ER and Golgi and perhaps of lysosomes (GERL-system) is
affected. This would be in agreement with the fact that in wobbler mice the vesicular
transport factor VPS54, a component of the GARP complex, is mutated [6]. In particular, vacuolisation in close proximity to the Golgi apparatus seen on electron
micrographs of wobbler motor neurons reflects the function of the GARP complex involved
in tethering endosome-derived retrograde vesicles to the Golgi membrane as shown in
yeast [7], but also in mammalian cells [14].

Immunofluorescence data indicate an intracellular accumulation of APP in Rab7- but
not LC3-positive endosomal compartments in degenerating wobbler motor neurons. Intracellular
APP accumulations have earlier been reported for Niemann Pick Disease type 1 (NPC1),
where APP was found in Rab5-positive early endosomes rather than in Rab7 positive
late endosomes [2]. Furthermore, Jin et al. [2] found elevated, proteolytically processed APP (C99 and Aβ42) in NPC1 neurons. The
western blotting data suggest that in wobbler spinal cords APP is upregulated but
no indications for abnormal APP proteolysis have been found indicating potential amyloid
accumulations. Thus, we conclude that the wobbler vesicle trafficking defect leads
to an accumulation of native APP variants in abnormally enlarged APP+/Rab7+ endosomal compartments.

The average cross-sectional diameter of these compartments is 3 μm and significantly
larger than APP- and Rab7-positive structures in wild-type or SOD1 transgenic mice.
The ultrastructural equivalents are vacuoles with low electron density and a size
of up to 3 μm. These vacuoles most likely are derived from the Golgi and the ER. Since
the occurrence of such structures is found in wobbler, but not in SOD1 and wild type
mice these are likely caused by impaired bidirectional vesicle traffic between ER
and Golgi. Again, these findings are in agreement with the function of Vps54 and the
GARP complex as recently demonstrated [14].

Additional File 1.APP/Rab7 as a diagnostic marker for impaired vesicle trafficking in MDF motor neurons. Immunohistochemical staining for Amyloid-Precursor Protein (APP,A) using rabbit
anti-APP, and for Rab7 (B) using goat-anti-Rab7, and co-localisation of both proteins
(C) in spinal cord motor neurons of 60 d.p.n. MDF mice. Note the large endosomal vesicles
(arrows in C) staining positive for APP and Rab7, indicating a transport impairment
in these motor neurons, similar to the ones observed in wobbler.

Extending our analysis to human MND were able to identify APP+/Rab7+ vesicles in degenerating motor neurons, which could indicate that in a subset of human
MND, retrograde vesicle trafficking is affected and could lead to the identification
of more MND relevant gene mutations. Thus the correlation between vesicle trafficking
defects and ALS is obvious since the affected gene for familial ALS2 Alsin was found
to encode a guanine-nucleotide exchange factor for the vesicle traffic associated
protein Rab5 [16,17] and ALS8 encodes the vesicle associated membrane protein B (VAPB, [18]). Besides these, several other vesicle trafficking proteins have been associated
with neurodegeneration like Dynactin with Neuropathy distal hereditary motor type
VIIB (HMN7B, [19] and Phosphoinositide phosphatase FIG4 in patients with ALS [20].

The VPS54 genes of some MND patients, preselected for enlarged APP- and LC3-positive
endosomal compartments were sequenced, but so far, mutations or polymorphisms were
not detected. A similar sequencing effort, although without pre-selection for APP
storage, has recently been published [21]. In this report a polymorphism in VPS54 has been identified but could not be directly
associated with the disease. Thus up to now, mutations or polymorphisms in VPS54 are
not defined as a major cause or a risk factor for sporadic forms of MND or ALS.

Conclusions

Our present study indicates that impaired vesicle trafficking in MND mouse mutants
is reflected by APP/Rab7 accumulation in spinal cord motor neurons. Some sporadic
forms of human MND or ALS show similar observations, so that assessment of APP localisation
in motor neurons could allow classification of human aetiologies with impaired vesicle
trafficking in order to identify additional MND-relevant genes.

Methods

Mice

Breeding and genotyping of C57Bl/6J-wr and C57Bl/6J stock mice was described earlier [6]. The SOD1 G93A transgenic mice were obtained from M. Sendtner (Würzburg University,
Germany) and genotyped for the SOD1 (G93A) transgene as described earlier [22]. Animal experimentation was done in accordance with the German law for the protection
of animals (TschG) and with a permit by the local authorities.

Human samples

Human MND and control autopsy samples were derived from the MRC London Neurodegenerative
Diseases Brain Bank (Institute of Psychiatry, King's College London) with an ethical
approval from local authorities. The case details including post mortem time before
formalin fixation or freezing of samples are provided in table 1. Generally, post-mortem time had no effect on the formation of APP-positive vesicles,
as spinal cord tissues from wild-type mice were used as controls 120 hours post-mortem
time and no changes in vesicular APP was observed (data not shown). Formalin fixed
tissues were embedded in paraffin according to standard procedures.

For immunohistochemistry, anti-APP directed against the extracellular domain (aa 301-315
of human APP, Sigma A8842, 1:500) was used in conjunction with anti-rabbit-Alexa 488
(1:500, MoBiTec); detection of the endosomal protein Rab7 was performed using goat-anti-Rab7
(SC-6563, Santa Cruz, 1:100) and mouse anti-goat-Cy3 (Dianova, Hamburg, 1:300). Both
primary antibodies were used simultaneously in a single-label experiment to avoid
cross-reactivity. For autophagosome detection, anti-LC3 antibody (1:200, Nanotools,
Munich) was used in conjunction with an anti-mouse Cy3 secondary antibody (1:1000,
Sigma) and anti-APP described above in a simultaneous incubation.

Fluorescence microscopy, confocal microscopy

Confocal images were obtained using Leica SP2 Confocal Laser Scanning Microscope with
software version 2.61 build 1537. Images were taken using either HCX PL APO CS 63.0
× 1.32 OIL UV or HC PL APO CS 20.0 × 0.70 IMM/COR objectives. To acquire different
fluorescence channels settings were optimized to prevent cross-talk, although images
were sequentially scanned to avoid this problem. All images were taken with 4 × line
average at data acquisition. Images were merged, and contrast and brightness was enhanced
using Leica software. Processing of image stacks was done with Volocity software.

Electron Microscopy

Mice were anaesthetized and transcardially perfused (with a permit from the local
authorities) in a 3 step procedure slightly modified according to Forssmann et al
[23] with 3% paraformaldehyde, 3% glutaraldehyde, 0,5% picric acid in 0.1 M sodium phosphate
buffer, pH 7.4 for 10 minutes. The spinal cord was dissected and fixed in the same
solution for additional 1-2 hours at 4°C, postfixed in 2% osmium tetroxide (2 h, 4°C)
and embedded in Araldite resin. For light microscopic identification, sections of
1.5 μm thickness were stained with Richardson's blue (1% w/v methylene blue, 1% w/v
Azur II) for 1 min, 80°C and visualised with a 100 × objective at a Zeiss Axiophot
microscope. For electron microscopy, 60-80 nm sections (stained for 40 min in uranyl
acetate and 7 min in lead citrate) were used (Zeiss EM 109).

Size determination of endosomal compartment

For quantification of vesicle areas we used the ImageJ program http://rsb.info.nih.gov/ij/download.htmlwebcite. Images were imported into image J, the scale was set to a defined length for normalisation.
Vesicles on the images were determined for cross-sectional diameters [μm]. Data were
obtained from a total of 100 motor neurons from 10 spinal cord sections per individual
(n = 10 per genotype, age-matched 42 d.p.n.) and are presented as mean ± SD. Significance
was determined using a paired t-test and values of p < 0.01 were considered significant.

Authors' contributions

All authors read and approved the final manuscript. RP performed the confocal microscopy
work, PG carried out the Nissl and immunostains and the western blots, PH did the
work associated with electron microscopy, CS has given intellectual input and sorted
out the human MND database, CT collected and delivered the human MND samples, TSJ
and JWB did the conceptual work on this manuscript including experimental planning
and manuscript preparation.

Acknowledgements

We thank Swetlana Schitek for immunohistochemical analyses, Trine Lund Petersen and
Edward Crutcher for expert technical assistance. Work was supported by the ALS Association
USA, by the King's College London (PhD studentship to P.G), Lundbeck Foundation, and
the Danish Research Council.